Antifungal substances and process for their production

ABSTRACT

There is disclosed a series of macrolides isolated from the fermentation broth of a microorganism identified as MA-5000 which morphological analysis reveals to be a strain of Streptomyces hygroscopicus. The compound&#39;s structure is presented based upon analytical studies. The compound has antifungal activity.

SUMMARY OF THE INVENTION

This invention is concerned with novel organic chemical compounds. Inparticular, it is concerned with phosphate esters which are produced bythe fermentation of a nutrient medium with a strain of the microorganismStreptomyces hygroscopicus MA-5000. Thus, it is an object of thisinvention to provide for such novel compound, and a method for preparingsuch product microbiologically. It is a further object of this inventionto provide for the recovery and purification of such compounds from thefermentation broth. These substances have antifungal activity, and itis, thus, an additional object of this invention to provide for novelantifungal compositions containing the disclosed compounds. Furtherobjects of this invention will become apparent from the followingdescription of this invention.

DESCRIPTION OF THE INVENTION

In accordance with this invention, a series of novel substances isdescribed, which are prepared by growing under controlled conditions, apreviously undescribed strain of a microorganism. A single novelcompound is produced by Streptomyces hygroscopicus MA-5000. The compoundis obtained by fermentation and recovered in substantially pure form asdescribed herein.

Based on taxonomic studies, the microorganism capable of producing thesecompounds is of a new strain of the microorganism Streptomyceshygroscopicus. One such culture, isolated from soil, is designatedMA-5000 in the culture collection of Merck & Co., Inc., Rahway, N.J. Asample of this culture, capable of producing the herein describedcompound, has been deposited, without restriction as to availability, inthe permanent culture collection of the American Type Culture Collectionat 12301 Parklawn Drive, Rockville, Md. 20852, and has been assigned theaccession number ATCC 39476.

The morphological and cultural characteristics of Streptomyceshygroscopicus MA-5000 are set forth below:

CULTURAL CHARACTERISTIC OF: Streptomyces hygroscopicus MA-5000-ATCC39476 (V-vegetative growth; A=aerial mycelium; SP=soluble pigment)

Morphology: Sporophores form compact spirals along aerial hyphae. Asculture ages, spore chains coalesce to form dark moist areas. Sporesurface by electron microscope (TEM) shows a smooth surface on somespores and a rough to warty surface on others.

Oatmeal agar (ISP Medium 3)

V: Reverse--brown edged with dark brown

A: Dark gray mixed with white and light gray, becoming black and moistwith age.

SP: Lt. grayish-brown.

Czapek Dox agar (sucrose nitrate agar)

V: Reverse--dark brown

A: moderate, grayish white

SP: Lt. brown

Egg albumin agar

V: Flat, spreading, grayish-brown

A: Light gray mixed with white and edged with dark gray, some areasbecoming moist and black.

SP: Light brown

Glycerol asparagine agar (ISP Medium 5)

V: Reverse--dark yellow brown

A: Light gray mixed with white and yellowish white. Droplets of goldenyellow exudate are present.

SP: Lt. tan

Inorganic salts-starch agar (ISP Medium 4 )

V: Reverse--light gray-brown edged with dark gray

A: Dark gray mixed with light gray and white, becoming black and moist.

SP: Light grayish-brown

Yeast extract-malt extract agar (ISP Medium 2)

V: Reverse--dark yellow brown

A: Dark gray becoming black and moist with age. Droplets of goldenyellow exudate also present.

SP: Light brown

Peptone-iron-yeast extract agar

V: tan

A: sparse, whitish

SP: None

Melanin: negative

Nutrient tyrosin agar

V: Rev.--brown

A: Moderate, light gray

SP: Light brown

Tyrosine Agar (ISP Medium 7)

V: Reverse--dark brown

A: Gray edged with dark gray; droplets of golden yellow exudate present

SP: Light brown

Carbon utilization

Pridham-Gottlieb basal medium+1% carbon source;

+=growth; ±=growth poor or questionable;

-=no growth as compared to negative control (no carbon source)

    ______________________________________                                        Glucose        +                                                              Arabinose      ±                                                           Cellulose      --                                                             Fructose       +                                                              Inositol       +                                                              Lactose        ±                                                           Maltose        +                                                              Mannitol       +                                                              Mannose        +                                                              Raffinose      ±                                                           Rhamnose       ±                                                           Sucrose        +                                                              Xylose         ±                                                           Temperature range                                                                            (Yeast extract-dextrose +                                                     salts agar)                                                    26° C. - Good vegetative growth and sporulation                        37° C. - Good vegetative growth scant aerial                           mycelia                                                                       50° C. - No growth                                                     ______________________________________                                    

Oxygen requirement (Stab culture in yeast extract-dextrose+salts agar)

Aerobic

All readings taken after three weeks at 28° C. unless noted otherwise.pH of all media approximately neutral (6.8-7.2)

A careful comparison of the foregoing data with published descriptions,including Bergey's Manual of Determinative Bacteriology 8th ed (1974);Waksman, The Actinomycetes Vol. II (1961); International Journal ofSystematic Bacteriology 18, 68-189, 279-392 (1968); 19, 391-512 (1969);22, 265-394 (1972); shows a close correlation between the description ofa bacterium identified as Streptomyces hygroscopicus and themorphological and cultural characteristics of MA-5285. It is thereforedetermined that MA-5285 is a new strain of the known speciesStreptomyces hygroscopicus.

The above description is illustrative of a strain of Streptomyceshygroscopicus MA-5000 which can be employed in the production of theinstant compound. However, the present invention also embraces mutantsof the above described microorganism. For example, those mutants whichare obtained by natural selection or those produced by mutating agentsincluding ionizing radiation such as ultraviolet irradiation, orchemical mutagens such as nitrosoguanidine or the like treatments arealso included within the ambit of this invention.

The instant compound is produced during the aerobic fermentation ofsuitable aqueous nutrient media under conditions described hereinafter,with a producing strain of Streptomyces hygroscopicus MA-5000. Aqueousmedia such as those used for the production of many antibioticsubstances are suitable for use in this process for the production ofthis macrocyclic compound.

Such nutrient media contain sources of carbon and nitrogen assimilableby the microorganism and generally low levels of inorganic salts. Inaddition, the fermentation media may contain traces of metals necessaryfor the growth of the microorganisms, and production of the desiredcompound. These are usually present in sufficient concentrations in thecomplex sources of carbon and nitrogen, which may be used as nutrientsources, but can, of course, be added separately to the medium ifdesired.

In general, carbohydrates such as sugars, for example dextrose, sucrose,maltose, lactose, dextran, cerelose, corn meal, oat flour, and the like,and starches are suitable sources of assimilable carbon in the nutrientmedia. The exact quantity of the carbon source which is utilized in themedium will depend, in part, upon the other ingredients in the medium,but it is usually found that an amount of carbohydrate between 0.5 and5% by weight of the medium is satisfactory. These carbon sources can beused individually or several such carbon sources may be combined in thesame medium.

Various nitrogen sources such as yeast hydrolysates, yeast autolysates,yeast cells, tomato paste, corn meal, oat flour, soybean meal, caesinhydrolysates, yeast extracts, corn steep liquors, distillers solubles,cottonseed meal, meat extract and the like, are readily assimilable byStreptomyces hygroscopicus MA-5000 in the production of the instantcompound. The various sources of nitrogen can be used alone or incombination in amounts ranging from 0.2 to 6% by weight of the medium.

Among the nutrient inorganic salts, which can be incorporated in theculture media are the customary salts capable of yielding sodium,potassium, magnesium, ammonium, calcium, phosphate, sulfate, chloride,carbonate, and like ions. Also included are trace metals such as cobalt,manganese, and the like. For the best production of the instantcompound, the addition of calcium carbonate to the production medium ismost preferred.

It should be noted that the media described hereinbelow and in theExamples are merely illustrative of the wide variety of media, which maybe employed, and are not intended to be limitative.

The following are Examples of media suitable for growing strains ofStreptomyces hygroscopicus MA-5000.

    ______________________________________                                        Medium A                                                                      Dextrose                  1.0    g.                                           Soluble starch            10.0   g.                                           Beef extract              3.0    g.                                           Yeast autolysate (As ardamine                                                                           5.0    g.                                           pH available from Yeast                                                       Products Inc., Clifton, N.J.)                                                 NZ Amine-E (caesin hydrolysate-                                                                         5.0    g.                                           available from Humko-Sheffield-                                               Memphis, Tenn.)                                                               MgSO.sub.4.7H.sub.2 O     0.05   g.                                           Phosphate Buffer          2.0    ml                                           CaCO.sub.3                0.5    g.                                           Distilled water           1000   ml.                                          pH                        7.0-7.2                                             Phosphate Buffer                                                              KH.sub.2 PO.sub.4         91.0   g                                            Na.sub.2 HPO.sub.4        95.0   g                                            Distilled water           1000   ml                                           pH                        7.0                                                 Medium B                                                                      Tomato paste              20.0   g.                                           Primary yeast             10.0   g.                                           Dextrin (CPC starch)      20.0   g.                                           CoCl.sub.2.6H.sub.2 O     0.005  g.                                           Distilled water           1000   ml.                                          pH                        7.2-7.4                                             Medium C                                                                      Corn meal                 20.0   g.                                           Distillers solubles       10.0   g.                                           Soybean meal              15.0   g.                                           Sodium citrate            4.0    g.                                           CaCl.sub.2.2H.sub.2 O     0.5    g.                                           MgSO.sub.4.7H.sub.2 O     0.1    g.                                           CoCl.sub.2.6H.sub.2 O     0.01   g.                                           FeSO.sub.4.2H.sub.2 O     0.01   g.                                           Polyglycol P2000 (Polypropylene glycol                                                                  2.5    mg.                                          mw 2000)                                                                      Distilled water           1000   ml.                                          pH                                6.5                                         Medium D                                                                      Lactose                   20.0   g.                                           Distillers solubles       15.0   g.                                           Autolyzed yeast (Ardamine pH)                                                                           5.0    g.                                           Distilled water q.s. to   1000   ml.                                          pH                        7.0                                                 Medium E                                                                      Tomato paste              40.0   g.                                           Oat flour                 10.0   g.                                           Distilled water           1000   ml.                                          pH                        7.0                                                 Medium F                                                                      Corn Steep Liquor         15.0   g.                                           (NH.sub.4).sub.2 SO.sub.4 4.0    g.                                           CaCO.sub.3                6.0    g.                                           Soluble Starch            20.0   g.                                           Corn meal                 1.0    g.                                           Soybean meal              4.0    g.                                           Glucose                   5.0    g.                                           KH.sub.2 PO.sub.4         0.3    g.                                           Lard oil                  2.5    g.                                           Distilled water           1000   ml.                                          pH                        6.7                                                 Medium G                                                                      Dextrose                  10.0   g                                            Asparagine                1.0    g                                            K.sub.2 HPO.sub.4         0.1    g                                            MgSO.sub.4.7H.sub.2 O     0.5    g                                            Yeast Extract             0.5    g                                            Oat Flour                 10.0   g                                            CaCO.sub.3                3.0    g                                            Trace Element Mix         10.0   ml                                           Distilled water           1000   ml                                           Adjust pH to 7.2                                                              Trace Element Mix                                                             FeSO.sub.4.7H.sub.2 O     1000   mg                                           MnSO.sub.4.4H.sub.2 O     1000   mg                                           CuCl.sub.2.2H.sub.2 O     25     mg                                           CaCl.sub.2.2H.sub.2 O     100    mg                                           H.sub.3 BO.sub.3          56     mg                                           (NH.sub.4).sub.6 MO.sub.4 O.sub.24.6H.sub.2 O                                                           19     mg                                           ZnSO.sub.4.7H.sub.2 O     200    mg                                           Distilled water           1000   ml                                           Medium H                                                                      Medium G                  1000   ml                                           Oat Flour                 10     g                                            pH                        7.2                                                 ______________________________________                                    

The fermentation employing Streptomyces hygroscopicus MA-5000 can beconducted at temperatures ranging from about 20° C. to about 40° C. Foroptimum results, it is most convenient to conduct these fermentations ata temperature in the range of from about 24° C. to about 30° C.Temperatures of about 27°-28° C. are most preferred. The pH of thenutrient medium suitable for producing the instant compounds can varyfrom about 5.0 to 8.5 with a preferred range of from about 6.0 to 7.5.

Small scale fermentations are conveniently carried out by placingsuitable quantities of nutrient medium in a flask employing knownsterile techniques, inoculating the flask with either spores orvegetative cellular growth of Streptomyces hygroscopicus MA-5000 looselystoppering the flask with cotton and permitting the fermentation toproceed in a constant room temperature of about 28° C. on a rotaryshaker at from 95 to 300 rpm for about 2 to 10 days. For larger scalework, it is preferable to conduct the fermentation in suitable tanksprovided with an agitator and a means of aerating the fermentationmedium. The nutrient medium is made up in the tank and aftersterilization is inoculated with a source of vegetative cellular growthof Streptomyces hygroscopicus MA-5000. The fermentation is allowed tocontinue for from 1 to 8 days while agitating and/or aerating thenutrient medium at a temperature in the range of from about 24° to 37°C. The degree of aeration is dependent upon several factors such as thesize of the fermentor, agitation speed, and the like. Generally thelarger scale fermentations are agitated at about 95 to 300 RPM and about2 to 20 cubic feet per minute (CFM) of air.

The novel compound of this invention is found primarily in the myceliumon termination of the Streptomyces hygroscopicus MA-5000 fermentationand may be removed and separated therefrom as described below.

The separation of the novel compound from the whole fermentation brothand the recovery of said compounds is carried out by solvent extractionand application of chromatographic fractionations with variouschromatographic techniques and solvent systems.

The instant compound has slight solubility in water, but is soluble inorganic solvents. This property may be conveniently employed to recoverthe compound from the fermentation broth. Thus, in one recovery method,the whole fermentation broth is combined with approximately an equalvolume of an organic solvent. While any organic solvent may be employed,it is preferable to use a water immiscible solvent such as ethylacetate, methylene chloride, chloroform and the like. Generally severalextractions are desirable to achieve maximum recovery. The solventremoves the instant compound as well as other substances lacking theantifungal activity of the instant compound. If the solvent is a waterimmiscible one, the layers are separated and the organic solvent isevaporated under reduced pressure. The residue is placed onto achromatography column containing preferably, silica gel. The columnretains the desired products and some impurities, but lets many of theimpurities, particularly the non-polar impurities, pass through. Thecolumn is washed with a moderately polar organic solvent such asmethylene chloride or chloroform to further remove impurities, and isthen washed with a mixture of methylene chloride or chloroform and anorganic solvent of which acetone, methanol, and ethanol and the like arepreferred. The solvent is evaporated and the residue furtherchromatographed using column chromatography, thin layer chromatography,preparative layer chromatography, high pressure liquid chromatographyand the like, with silica gel, aluminum oxide, ion exchange resins,dextran gels and the like, as the chromatographic medium, with varioussolvents and combinations of solvents as the eluent. Thin layer, highpressure, liquid and preparative layer chromatography may be employed todetect the presence of, and to isolate the instant compound. The use ofthe foregoing techniques as well as others known to those skilled in theart, will afford purified compositions containing the instant compound.The presence of the desired compound is determined by analyzing thevarius chromatographic fractions for physico-chemical characteristics.The structures of the instant compound have been determined by detailedanalysis of the various spectral characteristics of the compounds, inparticular their nuclear magnetic resonance, mass, ultraviolet andinfrared spectra.

The mass spectral data were obtained on the trimethylsilyl derivativesof the instant compounds, prepared by treatment withbis(trimethylsilyl)-trifluoroacetamide (BTSFA), 10:1 (v/v) in pyridineunder nitrogen at room temperature for 18 hours to afford molecular ionsin their electron impact mass spectra. Analagous silylation withperdeutero-BTSFA allowed unambiguous determination of the number ofsilyl groups in each molecule. High resolution exact mass measurementsindicated the following molecular formulae. (The underivatized nominalmolecular weights and molecular formulae have been included in the tablefor clarity).

    __________________________________________________________________________    Com-                                                                              Neat                                                                              Molecular                                                                            Corresponding                                                                         Silyl                                                                              Silyl                                             pound                                                                             Weight                                                                            Formula                                                                              Calculated                                                                            Found                                                                              Derivative                                        __________________________________________________________________________    I   599 C.sub.29 H.sub.46 NO.sub.10 P                                                        959.4836                                                                              959.4847                                                                           C.sub.29 H.sub.46 NO.sub.10 P + (C.sub.3                                      H.sub.8 Si).sub.5                                 II  513 C.sub.25 H.sub.40 NO.sub.8 P                                                         873.4468                                                                              873.4452                                                                           C.sub.25 H.sub.40 NO.sub.8 P + (C.sub.3                                       H.sub.8 Si).sub.5                                 III 613 C.sub.30 H.sub.48 NO.sub.10 P                                                        973.4992                                                                              973.4993                                                                           C.sub.30 H.sub.48 NO.sub.10 P + (C.sub.3                                      H.sub.8 Si).sub.5                                 IV  614 C.sub.30 H.sub.47 O.sub.11 P                                                         974.4832                                                                              974.4766                                                                           C.sub.30 H.sub.47 O.sub.11 P + (C.sub.3                                       H.sub.8 Si).sub.5                                 __________________________________________________________________________

The nuclear magnetic resonance spectrum for each of Compounds I, II, IIIand IV are shown in attached FIGS. 1 to 4 respectively. The spectra forCompounds I, III and IV were recorded in CD₃ OD at 60° C. and that forCompound II in CD₃ OD at 25° C. A small amount of ND₄ OD was added toCompound II to effect complete dissolution. Chemical shifts are shown inppm relative to internal tetramethylsilane (TMS) at 0 ppm.

Based on these experimental data, the instant compounds are believed tohave the following structural formula: ##STR1## wherein the compoundshave the following values for R₁, R₂ and R₃ :

    ______________________________________                                        R.sub.1          R.sub.2  R.sub.3                                             ______________________________________                                               ##STR2##      NH.sub.3.sup.⊕                                                                     OPO.sub.3 H.sup.⊖                       II    H              NH.sub.3.sup.⊕                                                                     OPO.sub.3 H.sup.⊖                       III                                                                                  ##STR3##      NH.sub.3.sup.⊕                                                                     OPO.sub.3 H.sup.⊖                       IV                                                                                   ##STR4##      OH       OPO.sub.3 H.sub.2                               ______________________________________                                    

Fields of technology adversely affected by the lack of effectivefungicides are many and include the paint, wood, textile, cosmetic,leather, tobacco, fur, rope, paper, pulp, plastics, fuel, rubber andfood industries. Fungicides are also utilized for agriculturalapplication, for instance in preventing or minimizing fungus growth onplants, fruits, seeds or soil.

Although many antifungal agents have been described and used heretoforein an effort to control fungi, none are entirely satisfactory andcontinued losses resulting from fungal attack make the problem ofcontrol a serious and lasting one. The number of fungicides practicallyuseful in combatting fungal growth have been small and only in a fewcases have synthetic organic chemicals been found applicable.

The compounds of the invention are effective in controlling the growthof Aspergillus species, for example A. niger, A. flavus, A. fumigatus,A. oryzae, A. luchensis, A. versicolor, A. sydowi, A. nidulans, A.flaucus and A. terreus, Penicillium species, for example, P. notatum, P.roqueforti, P. chrysogenum, P. oxalicum, P. spinulosum, P. martensii, P.citrinium, P. digitatum, P. expansum, P. italicum, P. cyclopium, and P.funiculosum, Neurospora series such as N. sitophila, Phoma species suchas P. terrestrius, Rhizopus species, Alternaria species such as A.solani, Chaetomium species such as C. globosum, Chaetomicum species, forexample, C. clivaceum, and Monilia species such as M. sitophila and M.nigra. The above fungi may be found on cosmetics, leather, electricalinsulation, textiles, and numerous other materials capable of supportingtheir growth.

The compounds of this invention may be employed in treatment of plants,soils, fruits, seeds, fur, wood and the like. The fungicidaleffectiveness of these compounds has been demonstrated against soilfungi, such as Rhizoctonia solani, Fusarium solani, and Pythium ultimum,plant fungi, for instance Erysiphe polygoni and Alternaria solani aswell as against saprophytes known to attack wood, pulp and lumber suchas Lenzites trabea and Ceratocystis pilifera and the fungus Pullulariapullulans which attacks paint.

In particular the compounds of this invention are useful in controllingthose agricultural fungus infections that attack growing plants. Thecompounds are particularly effective against those fungi, which causerice blast, tomato late blight, tomato early blight, wheat leaf rust,bean powdery mildew and tomato Fusarium wilt.

It should be understood that the compounds may be utilized in diverseformulations, solid, including finely divided powders and granularmaterials as well as liquid, such as solutions, emulsions, suspensions,concentrates, emulsifiable concentrate, slurries and the like, dependingupon the application intended and the formulation media desired.

Thus it will be appreciated that compounds of this invention may beemployed to form fungicidally active compositions containing suchcompounds as essentially active ingredients thereof, which compositionsmay also include finely divided dry or liquid diluents, extenders,fillers, conditioners and excipients, including various clays,diatomaceous earth, talc, and the like, or water and various organicliquids such as lower alkanols, for example ethanol and isopropanol, orkerosene, benzene, toluene and other petroleum distillate fractions ormixtures thereof.

When the active agents are employed in preventing topical fungal growth,one or more of the compounds may be uniformly distributed in a vehiclethat is chemically compatible with the particular compound selected,noninhibiting with respect to the action of the antifungal andessentially non-injurious to body tissue under the conditions of use.

It should be understood that the compounds of the invention may be usedin cmbination one with the other as well as with other fungicidallyactive materials. For instance, a mixture of the active compounds with2-(4'-thiazolyl)benzimidazole sorbic acid or its salts, propionic acidor its salts, mycostatin, sodium diacetate, trichomycin, amphotericin,griseofulvin, undecylenic acid, chlorquinadol,5,7-dichloro-8-hydroxyquinoline (Vioform), sodium o-phenylphenate,o-phenylphenol, biphenyl, chlorinated phenols, sodium benzoate,dehydroacetic acid and its salts or esters of parahydroxybenzoic acid,such as the methyl and propyl ester (parabens) can be used to givefungicidal effect when used in appropriate concentrations. It is quiteclear, too, that the compounds defined above may be used in conjunctionwith effective antibacterial materials in appropriate instances so as tocombine the action of each in such a situation as to be particularlyuseful, for instance, in applications where the presence of bacteriacreates undesirable results alongside the detrimental action of fungi.

It has been found that growth of various fungi existing in soil islimited or terminated by the addition to the soil of minor quantities ofthe compounds described. The term soil as used herein is intended toinclude all media capable of supporting the growth of plants and mayinclude humus, sand, manure, compost, artifically created plant growthsolution, and the like.

We have also found that the fungicides of the invention are effectiveagainst fungal diseases of plants, and may be effectively used either bydirect contact with the foliage or systemically, by introduction throughthe roots.

The following examples are being provided in order that the instantinvention may be more fully understood. Such examples are not to beconstrued as being limitative of the invention.

EXAMPLE 1

1. "A" Stage:

Culture MA5000 is maintained in the lyophilized state in a 1.0 ml ampulecontaining 0.15 ml of a skim milk suspension of the culture.

2. "B" Stage:

Vessel: 250 ml 3 baffled Erlenmeyer flask containing 50 ml medium perflask.

    ______________________________________                                                            Wt/Vol                                                    ______________________________________                                        Medium:                                                                       Dextrose              0.1%                                                    Soluble Starch        1.0%                                                    Beef Extract          0.3%                                                    Ardamine pH           0.5%                                                    NZ-Amine Type E       0.5%                                                    MgSO.sub.4.7H.sub.2 O 0.005%                                                  1.34 M Phosphate Buffer                                                                             0.02% Vol/Vol                                           CaCO.sub.3 (after pH adjustment)                                                                    0.05%                                                   Phosphate Buffer                                                              KH.sub.2 PO.sub.4     9.1%                                                    Na.sub.2 HPO.sub.4    9.5%                                                    ______________________________________                                    

Inoculum: Contents of one lyophilization tube into each "B" flask

Incubation: 48 hours at 28° C. on a rotary shaker with a 2" throwrotating at 220 RPM.

Sterility: Streak plates and gram stain

3. "C" Stage:

Vessel: 2-liter 3 baffled Erlenmeyer flask containing 500 ml medium

Medium: Same as "B" Stage

Inoculum: 10 ml from "B" Stage

Incubation: Same as "B" Stage

Sterility: Same as "B" Stage

4. "D" Stage:

Vessel: 189-liter stainless steel fermentor containing 160 liters ofmedium

    ______________________________________                                         Medium:                                                                      ______________________________________                                        Dextrose (Cerelose)    160    gms                                             Starch Modfied CPC     1600   gms                                             Meat Extract           480    gms                                             Ardamine pH            800    gms                                             NZ Amine Type E        800    gms                                             MgSO.sub.4.7H.sub. 2 O 8      gms                                             KH.sub.2 PO.sub.4      29     gms                                             Na.sub.2 HPO.sub.4     30     gms                                             pH to                  7.0-7.2                                                Add CaCO.sub.3         80     gms                                             ______________________________________                                    

Sterilization: 121° C. for 15 minutes

Inoculum: 500 ml from "C" Stage

Incubation:

(a) Temp: 28° C.

(b) Airflow: 3 ft³ /min

(c) Agitation: 150 RPM

(d) Pressure: 30 psig

(e) Cycle: 48 hrs

Defoamer: Polyglycol 2000

Sterility: Microscopic examination and YED streak plates at 28° C. and37° C.

5. "E" Stage:

Vessel: 756-liter fermentor containing 467 liters of medium

    ______________________________________                                        Medium:       Fermentation 1                                                                             Fermentation 2                                     ______________________________________                                        Corn Meal     9340    gms      9680  gms                                      Distillers Solubles                                                                         4670    gms      4840  gms                                      Soybean Meal  7005    gms      7260  gms                                      CaCl.sub.2.2H.sub.2 O                                                                       233.5   gms      242   gms                                      MgSO.sub.4.7H.sub.2 O                                                                       46.7    gms      48.4  gms                                      CoCl.sub.2.6H.sub.2 O                                                                       4.7     gms      4.8   gms                                      FeSO.sub.4.7H.sub.2 O                                                                       4.7     gms      4.8   gms                                      CaCO.sub.3    1868    gms      1936  gms                                      Polyglycol P-2000                                                                           116     ml       1.2   l                                        pH to         6.5              6.5                                            Volume        467     liters   484   liters                                   ______________________________________                                    

Sterilization: 121° C. for 15 minutes

Inoculum: 43 liters from "D" Stage

Incubation:

(a) Temp: 28° C.

(b) Airflow: 10 ft³ /min

(c) Agitation: 130 RPM

(d) Pressure: 13 psig

(e) Cycle: 3 days

Defoamer: Polyglycol P-2000

Sterility: Microscopic examination and YED streak plates at 28° C. and37° C.

Harvest: Into drums for work-up as in Examples 2 and 3

EXAMPLE 2

Four hundred liters of whole broth from Example 1 "E" Stage fermentation1, were filtered through a press after the addition of celite, a filteraid. The mycelial cake was extracted with 32 liters of 50% aqueousacetone for one hour. This slurry was filtered and the extractconcentrated in vacuo to an aqueous solution. This concentrate was addedto the filtered broth and the entire solution was extracted twice with ahalf volume of methylene chloride. The aqueous phase was then passedthrough 40 liters Dowex 1X2 (Cl⁻) column. After a water wash the columnwas eluted with 120 liters of 5% sodium chloride followed by 120 litersof 3% ammonium chloride in 90% methanol. The second eluate wasconcentrated in vacuo to 4 liters and eluted to 16 liters with water.This solution was passed through a 16 liter Amberlite XAD-2 column.After a water wash the column was eluted with 48 liters 10% aqueousacetone, 48 liters 20% aqueous acetone and 50% aqueous acetone. Thethird eluate was concentrated to an aqueous solution of 4 liters whichwas extracted twice with 8.8 liters butanol. The butanol extracts wereconcentrated to a small volume and the activity precipitated with theaddition of hexane. The precipitate was chromatographed on a 500 mlMallinkrodt CC-7 silica gel column in acetone with stepwise increases ofmethanol in acetone. Active cuts were combined and chromatographed on a225 ml LH-20 column in methanol. Active cuts were combined andrechromatographed on the same column in 25% acetone/methanol yielding549 mg. Aliquots of this purified preparation were chromatographed on apreparative RP-2, 10 μm HPLC column (9 mm×30 cm) using a methanol--1%HoAc gradient system. Fractions of the three separated components werecombined, concentrated to aqueous solutions and freeze-dried to yieldthe following samples:

Component I: 18 mg

Component II: 16 mg

Component III: 14 mg

The remainder of the LH-20 rich cut was chromatographed on a preparativeRP-18, 10 μm HPLC column (9 mm×30 cm) using the isocratic system500/450/50 methanol/water/1.0M PO₄ pH 6.5 to yield the followingsamples:

Component I: 15.8 mg

Component II: 9.9 mg

EXAMPLE 2

The fermentation of Example 1 using "E" Stage fermentation 2 was carriedout twice and each 400 liter fermentation was filtered 320 litersthrough a press after addition of celite. Each mycelial cake wasextracted with 320 liters of 50% aqueous acetone for one hour. Theacetone extracts were filtered and concentrated to aqueous solutions.These extracts and the filtrates were individually extracted with a halfvolume of methylene chloride and then each extracted twice with a halfvolume of butanol. The butanol extracts were combined and evaporatedonto 8 liters Grace silica gel. The coated gel was eluted sequentiallywith ethyl acetate, acetone, 10% methanol/acetone and 100% methanol. Themethanol eluate was concentrated (180 gm) onto 700 ml Baker silica geland placed on top of a two-liter silica gel column. This column waseluted with acetone, then stepwise with 20-40-75% methanol/acetone. Theactive cuts were concentrated, extracted with butanol (16 gm) andchromatographed on a two-liter EM 25-40 μm RP C-18 column with isocraticelution of 60/40 methanol/0.01M PO₄ pH 6.5. Cuts were combined based ontheir zone size and analytical HPLC analysis and rechromatographed on a200 ml EM 25-40 μm RP C-18 column using 55/45 methanol/buffer. Finalpurification of the individual active components was achieved bychromatography on a 10 μm RP C-18 column, 9 mm×50 cm.

Component II: 40 mg

Component III: 6.7 mg

Component IV: 2.5 mg

What is claimed is:
 1. A compound having the formula: ##STR5## whereinthe compounds have the following values for R₁, R₂ and R₃ :

    ______________________________________                                        R.sub.1          R.sub.2   R.sub.3                                            ______________________________________                                                ##STR6##     NH.sub.3.sup.⊕                                                                      OPO.sub.3 H.sup.⊖                      II     H             NH.sub.3.sup.⊕                                                                      OPO.sub.3 H.sup.⊖                      III                                                                                   ##STR7##     NH.sub.3.sup.⊕                                                                      OPO.sub.3 H.sup.⊖                      IV                                                                                    ##STR8##     OH        OPO.sub.3 H.sub.2                              ______________________________________                                    


2. The compound of claim 1 wherein R₁ is ##STR9## R₂ is --NH₃.sup.⊕ andR₃ is --O--PO₃ H.sup.⊖.
 3. The compound of claim 1 wherein R₁ is H, R₂is --NH₃.sup.⊕ and R₃ is --O--PO₃ H⊖.
 4. The compound of claim 1 whereinR₁ is ##STR10## R₂ is --NH₃.sup.⊕ and R₃ is --O--PO₃ H.sup.⊖.
 5. Thecompound of claim 1 wherein R₁ is ##STR11## R₂ is --OH and R₃ is--O--PO₃ H₂.
 6. An antifungal composition which comprises one or morecompounds of claim 1 and a pharmaceutically acceptable carrier.
 7. Amethod for the treatment of fungus infections which comprises treatingan area infected with fungi with one or more compounds of claim 1.